Antibodies are blood serum proteins, sometimes called immunoglobulins, which are generated by the immune system in response to a foreign substance or organism (antigens). Antibodies are characterized by specific binding with their particular antigen, neutralizing them so that they are removed from the circulation. Antibody technology is widely used for the diagnosis, monitoring, prevention and treatment of many different ailments.
Several types of immunoglobulins (Ig) have been identified, such as, IgG, IgM, IgD, IgA and IgE. The majority of commercially produced immunoglobulins are the IgG-type, because they constitute a large amount of the Ig in blood serum and are associated with a mature immune response. While all IgGs have the same general structure, they fall into different isotype categories, such as IgG1, IgG2, IgG3, and IgG4. IgGs are composed of four polypeptide chains, two that are heavy (H) and two light (L). Each heavy chain is linked to a light chain via a disulfide bond and the two heavy chains, in turn, are joined together by disulfide bridges at a region known as the hinge. Each heavy chain has three constant regions, CH1, CH2, and CH3, the last two in the carboxy terminal region (after the hinge) and the first in the amino terminal region (immediately before the hinge) and a Variable region (VH) in the amino terminal end, while each light chain has only one constant region, CL, in the carboxy terminal end and one variable region, VL, in the amino terminal end.
Enzymatic digestion of IgG can result in a number of different fragments, depending on the enzyme used. Papain and pepsin are among the most common enzymes used to digest IgGs. Papain typically generates three fragments, the crystallizing fragment (Fc) and two antigen-binding fragments (Fab). Pepsin typically generates one F(ab′)2 fragment and completely digests the Fc fragment. Fab and F(ab′)2 fragments can retain the capacity for specific binding to their specific antigen. F(ab′)2 also precipitates its specific antigen. The other antibody fraction, Fc, typically acts as a marker signal for macrophages and the activation of lymphocytes for the recognition and phagocytosis of the antigen-antibody complex.
The Fc fragment comprises the antigenic determinants of the antibody in such a way that, when a patient is administered whole antibodies generated in, e.g., an animal of another species, the patient may generate an immune response against these antigenic determinants. This can give rise to varied adverse secondary responses, including anaphylactic shock. These problems can be reduced by digesting the antibodies with enzymes such as papain or pepsin. These enzymes can generate Fab, F(ab′)2, and Fc fractions that can be isolated and purified, and allow for administration of Fab or F(ab′)2 fragments to a subject.
The F(ab′)2 fragment has a particular advantage over Fab in that it is retained in the organism for a longer period because of its molecular weight. Moreover, F(ab′)2 retains the capacity to precipitate its antigen under physiological conditions. As the F(ab′)2 fragment retains the specific binding character of the intact antibody, its utility is similar to the intact antibody. However because the F(ab′)2 fragment lacks the Fc fragment, it is less likely to be recognized as foreign by the recipient, thus providing greater tolerance to application and reducing the possibility of secondary reactions, which is particularly useful for prolonged treatments such as those applied in autoimmune diseases.
Many methods for the production of antibodies and their fragments are known. For example, in U.S. Pat. No. 4,849,352, Sullivan et al. describes the production of Fab fragments through the digestion of antibodies with papain immobilized in polyacrylamide, and F(ab′)2 fragments through the digestion of antibodies with immobilized pepsin. A number of these methods for the production of antibody fragments such as F(ab′)2, by means of digestion of whole antibodies with pepsin have shown several disadvantages, such as considerable loss of biological activity, a high residual content of whole antibodies and other impurities, and digestion of the F(ab′)2 fragment itself. Thus, straightforward methods are needed that allow for the production of F(ab′)2 fragments from whole antibodies and antibodies that comprise sequence in addition to the F(ab′)2 region, which methods provide high fragment yields and retains the binding activity of the intact antibody.